Design rules for selective binding of nuclear localization signals to minor site of importin α

Selectivity is a critical issue in molecular recognition. However, design rules that underlie selectivity are often not well understood. Here, we studied five classical nuclear localization signals (NLSs) that contain the motif KRx(W/F/Y)xxAF and selectively bind to the minor site of importin α. The selectivity for the minor site is dissected by building structural models for the NLS-importin α complexes and analyzing the positive design and negative design in the NLSs. In our models, the KR residues of the motif occupy the P1' and P2' pockets of importin α, respectively, forming hydrogen-bonding and cation-π interactions. The aromatic residue at the P4' position plays dual roles in the selectivity for the minor site: by forming π-stacking with W357 of importin α to reinforce the minor-site binding; and by clashing with the P5 pocket in the major binding site. The F residue at the P8' position occupies a deep pocket, providing additional stabilization. The P7' position sits on a saddle next to the P8' pocket and hence requires a small residue; the A residue fulfills this requirement. The principal ideas behind these blind predictions turn out to be correct in an evaluation against subsequently available X-ray structures for the NLS-importin α complexes, but some details are incorrect. These results illustrate that the selectivity for the minor site can be achieved via a variety of design rules.